Lactobacillus helveticus mutants having low increase in acidity of lactic acid during storage

ABSTRACT

Lactic acid bacteria of the genus Lactobacillus having the following specific properties: (1) an increased amount of acidity of lactic acid when storing a product cultured by the bacteria at 10° C. for two weeks being 0.5% or less; (2) an activity of cell membrane bound adenosine triphosphatase being 5 μmol·Pi/min/mg protein or less; and (3) the bacteria having neomycin resistance.

BACKGROUND OF THE INVENTION

This invention relates to a fermented milk product in which the increasein acidity in the low pH region and during storage at lower temperaturesis inhibited, and novel lactic acid bacteria of the genus Lactobacillusthat may be employed for the preparation of such fermented milk product.

The lactic acid bacteria of the genus Lactobacillus have long been knownas a representative starter for preparation of fermented milk. Thelactic acid bacteria of rod shape, such as the genus Lactobacillusproduces lactic acid of higher acidity than that of the lactic acidbacteria of coccus such as the genus Lactococcus or the genosStretococcus, while being frequently higher in its protease activity ofdecomposing milk protein. In particular, Lactobacillus helveticusexhibits strong protein-decomposing activity. The peptide generated bydecomposition of milk protein by its extracellular protease has beenreported to exhibit the inhibition activity against angiotensinconverting enzyme (Hereinafter referred to as ACE) which is a substanceresponsible for increase in blood pressure. Similar activity may benoticed with the fermented milk by the Lactobacillus helveticus. TheseACE inhibitory peptides have been confirmed to exhibit the activity inlowering the blood pressure with spontaneously hypertensive rats (SHR),as reported by Nakamura, Y. et al NIPPON NOGEI KAGAKU KAISHI, 67, 289,1993.

However, Lactobacillus helveticus produces a large quantity of lacticacid during milk fermentation and hence is lowered significantly in pH.Besides, the acidity of lactic acid tends to be increased during storageat low temperature. Consequently, Lactobacillus helveticus isdifficultly used for the preparation of yogurt, etc., such that it isscarcely used at present for yogurt-like fermented milk products.

In general, the increase in acidity during storage following thepreparation of yogurt poses a serious problem. For suppressing suchincrease in acidity, various trials have been made such as inprovementof strains by mutation. However, desired mutant strains have not beenproduced because mutant strains low in growth activity tend to beseparated.

Adenosine triphosphatase (Hereinafter referred to as ATPase) has so farbeen confirmed to be taking part in intracellular pH adjustment in themicroorganisms. Although the extracellular pH of lactic acid bacteria isin an acidic range by the production of lactic acid as a finalmetabolite in the fermentation of lactic acid bacteria, theintracellular pH of the lactic acid bacteria is maintained in a neutralrange by the action of ATPase. On the other hand, ATPase tends to beproduced in an increasingly larger quantity with increase in thedifference between the intracellular pH and the extracellular pH. Thus,it has been confirmed that, under the conditions in which such cell-wallbarrier is destroyed in the acidic medium to eliminate the protonicconcentration gradient, the microorganism cells cannot be proliferated(The Journal of Biological Chemistry, 261, 2.627-630, 1986, by HiroshiKobayashi, Takeshi Suzuki and Tsutomu Umemoto; and The Journal of DairyScience, 74, 747-751, 1991, by Nancy L-Nannen and Robert W. Hutkins).While, these reports are directed to the relation between theextracellular pH and ATPase activity of the cells, there is nodisclosure as to the final pH of a fermented product obtained byfermentation of the microorganisms.

Concerning neomycin resistance, the neomycin-resistant mutant strainEscherichia coli has been known to be devoid of ATPase activity (J. ofBacteriology, 116, 3. 1124-1129, 1973, by Barry P. Rosen). However,there is no disclosure as to lactic acid production.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide lactic acid bacteriaof the genus Lactobacillus which is a mutant strain having low increasein acidity of lactic acid during culturing and storage of th culturedproduct while maintaining characteristics of lactic acid bacteria of thegenus Lactobacillus such that it exhibits substrate specificity againstmilk protein and strong proteolytic activity and produces the ACEinhibitory peptide in the fermented milk.

It is another object of the present invention to provide a fermentedmilk product in which increase in acidity of lactic acid is suppressedin the low pH range and during storage at lower temperatures.

The above and other objects of the present invention will becomeapparent from the following description.

The present invention provides lactic acid bacteria of the genusLactobacillus having the following specific properties:

(1) an increased amount of acidity of lactic acid when storing a productcultured by the bacteria at 10° C. for two weeks being 0.5% or less;

(2) an activity of cell membrane bound adenosine triphosphatase being 5μmol·Pi/min/mg protein or less; and

(3) the bacteria having neomycin resistance.

The present invention also provides a fermented milk product produced byfermentation using the above-defined lactic acid bacteria.

PREFERRED EMBODIMENTS OF THE INVENTION

The present invention will be described in more detail hereinbelow.

The lactic acid bacteria of the genus Lactobacillus according to thepresent invention (hereinafter referred to simply as "lactic acidbacteria") is a mutant strain of the lactic acid bacteria of the genusLactobacillus. Although the lactic acid bacteria of the presentinvention is not changed in the microbiological properties owned by thelactic acid bacteria of the parent strain and is not changed above allin intensity of the extracellular protease activity and substratespecificity, it exhibits characteristics distinct from the parent strainin the following three points, namely, (1) an increased amount ofacidity of lactic acid when storing a product cultured by the bacteriaat 10° C. for two weeks being 0.5% or less; (2) an activity of cellmembrane bound adenosine triphosphatase being 5 μmol·Pi/min/mg proteinor less; and (3) the bacteria having neomycin resistance.

It suffices if the increased amount of acidity of lactic acid whenstoring a product cultured by the bacteria at 10° C. for to weeks is0.5% or less and preferably 0.4% or less, while there is no limitationto the increased amount of acidity of lactic acid under any otherconditions. However, the final acidity of he lactic acid at the time ofcultivation is preferably 0.5 to 1.2% and more preferably 0.6 to 1.0%,and when storing a product cultured by the bacteria at 10° C. for oneweek, the increase of acidity of lactic acid is preferably not higherthan 0.4% and more preferably not higher than 0.2%.

Although the activity of cell membrane bound ATPase is changed with thepH in the culture medium, it is not higher than 5 mol·Pi/min/mg protein.The cell membrane bound ATPase activity can measured by the method ofNeu, H. C & Heppel. L. A. J. Biol. Chem. 240. 3685 (1965), that is byextracting the protein under an osmotic shock, adding 40 μl of theextracted solution to 10 μl a of a solution (pH 6.5) of 250 mM oftris-hydrochloric acid, 25 mM of magnesium chloride, 25 mM of adenosinesodium triphosphate and 16.5 g/m bovine serum albumin (BSA), forreaction at 37° C. for one minute, adding 5 μl of 0.1N hydrochloric acidto the reaction solution to terminate the reaction, and subsequentlymeasuring the concentration of the produced inorganic phosphoric acidusing a reagent "P-TEST WAKO" produced by WAKO PURE CHEMICAL INDUSTRIES,LTD., and by the method proposed by Bradford (Anal. Biochem. 72, 284,1976, by Bradford M. M.) in which the protein concentration in theextracted solution is measured.

The lactic acid bacteria exhibits neomycin resistance. It suffices if itis capable of being proliferated without extinction when cultured on aneomycin-containing culture medium.

There is no limitation to the lactic acid bacteria of the presentinvention if it exhibits bacteriological properties of the parent strainand also exhibits the specific properties (1) to (3) as mentioned above.A specific example of such bacteria is the lactic acid bacteria of thegenus Lactobacillus deposited with the NATIONAL INSTITUTE OF BIOSCIENCEAND HUMAN TECHNOLOGY, AGENCY OF INDUSTRIAL SCIENCE AND TECHNOLOGY, asFERM-P13914 under the International Deposit No.BP-4835 (Hereinafterreferred to as CPN4).

The bacteriological properties of CPN4 were analyzed by the methodproposed by Mitsuoka (Journal of Medical Technology, 18.1163, 1974). Bythese analyses for identification, it could be confirmed that, while thebacteria CPN4 exhibited bacteriological properties similar to those ofthe parent strain Lactobacillus helveticus JCM 1004, it exhibitedproperties evidently different from those of the parent stain in pointof neomycin resistance, low ATPase activity and low growth rate in thelow pH range, so that it could be identified as being its pH-sensitivemutant strain. The specific bacteriological properties of the mutantstrain are given in the following Table 1:

TABLE 1

I. Morphological Properties

1) Morphology; rod shape

2) Mobility; none

3) Spore Formation; none

4) Gram Stain; positive

II. Physiological Properties:

1) Catalase Production; negative

2) Indole Production; negative

3) Nitrate Reduction; negative

4) Aerobic Growth; facultative anaerobic

5) Formation of DL-lactic acid from glucose by homolactic fermentationwithout formation of gases

6) Formation of Acids from Carbohydrates;

glucose; +

maltose; -

Lactose; +

cellobiose; -

mannose; +

trehalose; -

fructose; +

melibiose; -

galactose; +

raffinose; -

sucrose; -

stachyose; -

mannitol; -

arabinose; -

sorbitol; -

xylose; -

esculin; -

rhamnose; -

salicin; -

7) Neomycin resistance; growth in 40 μg of neomycin/ml or less

8) ATPase activity; 5 μmol·Pi/min/mg protein or less

The lactic acid bacteria of the present invention may be prepared bymutating the lactic acid bacteria of the genus Lactobacillus, i.e. theparent strain, by a method which will now be explained.

There is no limitation to the parent strain provided that it is of thegenus Lactobacillus. Examples of such parent strains include lactic acidbacteria having high lactic acid producibility, such as Lactobacillushelveticus or Lactobacillus delbuneckii subsp. bulgaricus.

The lactic acid bacteria of the genus Lactobacillus are proliferativelycultured on a culture medium exhibiting high proliferation for eachstrain, such as Briggs Liver Broth, MRS culture medium, BL culturemedium or GAM culture medium. The strains thus cultured are collected inthe log phase. The innoculum size of the lactic acid bacteria in eachculture medium is preferably about 1×10⁶ cells/ml. For culturing, it ispreferred that the culturing temperature be 30° to 45° C. and theculturing time period be 3 to 24 hours.

Since it is proposed by Barry (J. of Bacteriology, 116, 1124-1129, 1973,by Barry, P. R.) that screening from the neomycin resistant strains iseffective for isolating the strains with low ATPase activity, thecollected parent strains are inoculated on a proliferation agar mediumcontaining 5 to 100 μg/ml of neomycin for selecting neomycin resistantstrains in accordance with this proposal.

For inducing of the mutation effectively with the proposed method, theparent strains may be pre-treated by, for example UV irradiation or achemical mutagen. For UV irradiation, it is preferred that the UV raysbe not more than 500 nm in wavelength, the radiation time period beseveral seconds to 30 minutes and the irradiation distance be 10 to 30cm. For effective selection of the mutant strains, it is preferred thatviable bacteria in the log phase be suspended in the culture medium forproliferation and UV ray irradiation be conducted under the conditionwhich will give the viability of 0.1 to 1.0%. For processing by thechemical mutagen, such as nitrosoguanidine(N-methyl-N'-nitro-nitrosoguanidine), ethyl methane sulfonic acid,methyl methane sulfonic acid or 5-bromouracil are employed as thechemical mutagen. If nitrosoguanidine, for example, is employed, viablebacteria in the log phase are suspended in the culture medium forproliferation having the pH value of 5.0 to 8.0, preferably 6.0 to 7.0,and processing is preferably carried out at the processing temperatureof from room temperature to 50° C., above all, from 30° to 37° C., for aprocessing time of 10 to 60 minutes, above all, for 15 to 30 minuteswith the nitrosoguanidine concentration of 30 to 1000 g/m, above all,100 to 300 g/m.

After selecting the neomycin resistant strains, those strains showinglow proliferation are discarded and those showing low proliferation onlyat pH of 4.5 or lower are selected. The selected strains showing lowproliferation at the low pH range are cultured in a milk medium, such asskim milk medium, at 30 to 45° C. for 1 to 3 days, and those cellstrains having the final pH higher than that of the parent strains, thatis the cell strains having a low increase in the final acidity of lacticacid is selected. The mutant strains having the final pH following thecultivation at pH 3.4 to 3.8 are ultimately selected and cultured in amilk medium at e.g. 30 to 45° C. for 3 to 24 hours. The cell strainsultimately exhibiting the same bacteriological properties as the parentstrains and exhibiting the properties (1) to (3) are selected as thelactic acid bacteria of the present invention.

There is no particular limitation to the fermented milk product of thepresent invention provided that the product has been processed byfermentation using the above-mentioned lactic acid bacteria. Examples ofthe fermented milk products include yogurt, lactic acid fermentedbeverage or cheese, which may be produced by the conventional methods.The fermented milk product may be produced by employing any of knownlactic acid bacteria, such as Streptococcus thermophilus, Lactococcuslactis or Bifidobacterium longim in combination with the lactic acidbacteria of the invention. For preparing the fermented milk products,any of known methods for preparation may be employed, on the conditionthat the above-defined lactic acid bacteria are employed.

The lactic acid bacteria of the genus Lactobacillus of the presentinvention may be employed for the preparation of fermented milkproducts, such as yogurt, in which it is possible to suppress theincrease in the acidity of lactic acid on prolonged storage at lowertemperatures. The fermented milk product of the present invention,employing the above-defined lactic acid bacteria, exhibits theantihypertensive activty, while maintaining the flavor peculiar to theproduct directly following the end of fermentation, in addition toexhibiting the properties of suppressing the increase in the acidity oflactic acid on prolonged storage at lower temperatures.

EXAMPLES OF THE INVENTION

The present invention will be explained with reference to Examples whichare given for illustration only and are not intended for limiting theinvention.

Example 1 Isolation of Mutant Strains

1×10⁶ cells/ml of Lactobacillus helveticus JCM-1003, referred tohereinafter as the parent strain, were inoculated in the Briggs liverbroth, and cultured at 37° C. for 20 hours. The cells were re-culturedin a new culture medium of the same type at 37° C. for 6 hours and thebacteria in the log phase were collected and irradiated with UV raysusing a 15 W UV lamp for the irradiation time of 30 seconds at theirradiation distance of 20 cm. The bacteria were then inoculated on theBriggs agar culture medium containing 5 μg/ml of neomycin so that theviable bacteria were equal to 1×10⁶ cells, and were cultured at 37° C.for two days. Fifty strains each capable of forming a larger colony wereselected and replicated on a Briggs agar media adjusted to pH of 6.5 and4.5. Fifteen strains showing the proliferation comparable to that of theparent strain on the culture medium of pH 6.5 and showing theproliferation lower than that of the parent strain on the culture mediumof pH of 4.5 were selected and inoculated on a 9% skim milk. Theculturing was continued at 37° C. for three days and the final pH valuesof the cultured strains were compared to those of the parent strain. Thesole strain, referred to hereinafter as CPN4, having a high final pH ascompared to that of the parent strain was selected. The mutant strainCPN4 and the parent strain were cultured for three days and measurementwas made of the final pH and turbidity (absorbance at 590 nm). Theresults are shown in the following Table 2.

                  TABLE 2                                                         ______________________________________                                        Strains       Final pH  Turbidity (O.D. 590 nm)                               ______________________________________                                        L. helveticus JCM1003                                                                       3.23      3.58                                                  L. helveticus CPN4                                                                          3.64      2.54                                                  ______________________________________                                    

Measurement of Cell Membrane Bound ATPase Activity

Nine strains of the Lactobacillus helveticus were cultured in a Briggsculture medium, pH 5.0, at 37° C. for 6 hours. From each cell, proteinwas extracted under the osmotic shock in accordance with the Neu'smethod (J. Biol. Chem. 240 3685, 1965, by Neu, H. C. & Heppel. L. A.).40 μl of the extracted solution was added to 10 μl of a solutioncontaining 250 mM tris-hydrochloric acid, pH 6.5, 25 mM of magnesiumchloride, 25 mM of adenosine sodium triphosphate and 16.5 μg/ml ofbovine serum albumin (BSA) and the resulting mixture was reacted at 37°C. for 10 minutes. The reaction was terminated by adding 5 μl of 0.1Nhydrochloric acid to the reaction mass. The concentration of theproduced inorganic phosphoric acid was measured using "P-TEST WAKO", areagent manufactured by WAKO PURE CHEMICAL INDUSTRIES, LTD. The proteinconcentration in the extracted solution was measured by the Bradford'smethod (Anal. Biochem., 72 284, 1976, by Bradford M. M.). The ATPaseactivity was measured under the above-mentioned conditions andrepresented in terms of the amount yielded of the inorganic phosphoricacid by a unit amount of protein containing enzyme (mol·Pi/min/mgprotein) (Table 3). The neomycin resistance of each strain was found bymeasuring the minimum growth inhibition concentration. The results areshown in the following Table 3.

                  TABLE 3                                                         ______________________________________                                                     ATPase activity                                                                            Neomycin resistance                                              (pmol · Pi/                                                                       (minimum inhibition                                 Strains      min/mg protein)                                                                            concentration μg/ml)                             ______________________________________                                        L. helveticus CPN4                                                                         3.6          40                                                  L. helveticus JCM1003                                                                      22.0         10                                                  L. helveticus CP611                                                                        17.7         10                                                  L. helveticus CP615                                                                        15.6         10                                                  L. helveticus JCM1120                                                                      12.0          5                                                  L. helveticus JCM1004                                                                      13.1         10                                                  L. helveticus JCM1006                                                                      16.8         10                                                  L. helveticus JCM1007                                                                      15.2          5                                                  L. helveticus JCM1008                                                                      14.4          5                                                  ______________________________________                                    

From the results of Table 3, it was confirmed that the parent strainJCM-1003 and other seven strains showed high levels of ATPase activity.However, the CPN4 strain showed ATPase activity lower than that of theparent strains.

The CPN4 strain and the parent strains were again cultured in a 9% skimmilk at 37° C. for 30 hours, and measurement was made in accordance withthe following method, as to whether or not the ACE inhibition activityand the peptide quantity of these mutant strains in the fermented milkwere on the same order of magnitude as those of the parent strains. Forreference, the extracellular protease activity of each strain was alsomeasured and the final pH value was checked again. These results ofmeasurement are shown in Table 4.

Measurement of ACE Inhibition Activity in Fermented Milk

Measurement of the ACE inhibition activity was conducted in accordancewith the method by Cushman and Cheung (Pharmacol., 20 1637, 1971, byCushman D. W. & Cheung H. S.). Each fermented milk sample wascentrifuged at 15000 rpm for five minutes, to produce a supernatant(whey), which was then adjusted to a neutral pH using a 1N sodiumhydroxide solution. 80 μl of the supernatant was transferred to a testtube, to which was added 0.2 ml of hippuryl histidyl leucine(Hip-His-Leu produced by SIGMA CHEMICAL CO. ) adjusted to 5 mM with a0.1 M boric acid buffer containing 0.3 M NaCl pH 8.3 as a substrate. 20μl of an enzyme solution (0.1 unit/ml, produced by SIGMA CHEMICAL CO.)was added to the reaction system and the reaction was carried out at 37°C. for 30 minutes. 250 μl of 1N hydrochloric acid was then added to thereaction system to terminate the reaction. 1.7 m of ethyl acetate wasadded to the reaction system and stirred for about 20 seconds. Thereaction system was centrifuged at 3000 rpm for 10 minutes in order torecover 1.4 m of an ethyl acetate layer. The solvent was then removed byheating at 120° C. for 40 minutes. After removing the solvent, 1 m ofdistilled water was added and, after stirring for about 20 seconds,absorbance at 228 nm of the extracted hippuric acid was measured. 1 unitof the enzyme activity was calculated by the following equation as anamount which gave 50% inhibition of the ACE activity:

    Enzyme quantity (1 unit)=[(A-B)/(A-C)]×100×1/50

where A is the absorbance (228 nm) not containing the sample, B is theabsorbance (228 nm) admixed with the sample and C is the absorbance (228nm) not admixed with the enzyme nor the sample.

The peptide quantity was determined in accordance with the OPA method(J. of Dairy Science, 66 1219, 1983, by Charch F. C. et al.). Thestandard curve was prepared using a trypsin digested product of caseinas a standard.

Measurement of Extracellular Protease Activity

The extracellular protease activity was measured in accordance with themethod by Yamamoto et al. (J. of Biochem. 114, 740, 1993, by Yamamoto etal.), based upon the method by Twining S. (Anal. Biochem. 143 3410, byTwining. S.). That is, each strain was cultured by maintaining at pH 6.0in a 9% skim milk and bacteria thus produced were collected in themid-log phase. Sodium citrate was added so that the final concentrationwas 1% to render the milk culture medium to be transparent followed bycentrifugal separation at 5000 rpm for 10 minutes to collect thebacteria. The bacteria were washed with 50 mM β-glycerophosphoric acidand then suspended in a 50 mM tris-hydrochloric acid buffer (pH 7.8) andmeasurement was made of the enzymatic activity of the bacterial cellsurface. 30 μl of the liquid suspension of bacterial cell was added to20 μl of 0.4% fluoresceine-isothiocyanate-casein produced by SIGMACHEMICAL CO. After incubation at 42° C. for one hour, 120 μl of a 5%trichloroacetic acid solution was added to the reaction system. Afterallowed to stand for about 20 minutes at room temperature, the reactionsystem was centrifuged at 15000 rpm for ten minutes. 60 μl of thesupernatant was added to 3 ml of a 500 mM tris-hydrochloric acid buffer(pH 7.8) and fluorometry was carried out of the reaction mass. Forfluorometry, measurement was made of the fluorescence of 525 nm producedat an excitation wavelength of 490 nm. As for the enzymatic activity,the amount of enzyme corresponding to 1% intensity of fluorescence oftotal substrate was defined as being 1 unit. The CPN4 strain exhibitedextracellular protease activity comparable to that of the parent strain.Similar values were obtained of the peptide quantity and the ACEinhibition activity. However, the final pH was again confirmed to behigher than that of the parent strain, Consequential, only the CPN4strain showing the ACE inhibition activity in its fermented milkcomparable to that of the parent strain and having the final pH higherthan that of the parent strain were selected as being satisfactory.

                  TABLE 4                                                         ______________________________________                                                                       ACE    Protease                                                      Peptide  inhibition                                                                           activity                                                      quantity activity                                                                             (U/O.D.                                 Strains      Final pH (%)      (U/ml) 590 nm)                                 ______________________________________                                        L. helveticus JCM1003                                                                      3.24     0.24     32.3   101                                     L. helveticus CPN4                                                                         3.62     0.23     28.6   103                                     ______________________________________                                    

Low Temperature Storage Test

A low temperature storage test of the mutant strain CPN4 strain was thenconducted at 10° C. The parent strain and the CPN4 strain were culturedin milk at 37° C. and at a time point when the amount of the producedlactic acid reached 0.6%, the fermented milk was cooled and stored at10° C. In one week and in two weeks after storage, the pH value, theacidity of lactic acid (%), the ACE inhibition activity and the peptidequantity were measured. The results are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                                                       ACE                                                                  pH       inhibition                                                                           Peptide                                              Storage  Acidity  activity                                                                             quantity                                Strains      period   (%)      (U/ml) (%)                                     ______________________________________                                        L. helveticus JCM1003                                                                      Start of 4.3   0.68 19.2   0.14                                               storage                                                                       One      4.1   1.12 20.2   0.17                                               week                                                                          Two      3.7   1.27 26.2   0.17                                               weeks                                                            L. helveticus CPN4                                                                         Start of 4.3   0.68 17.7   0.16                                               storage                                                                       One      4.2   0.83 18.5   0.19                                               week                                                                          Two      4.1   0.95 23.3   0.20                                               weeks                                                            ______________________________________                                    

It is seen from the results of Table 5 that the fermented milk of theCPN4 strain showed no significant difference from that of the parentstrain in the peptide quantity and in ACE inhibition activity. With theparent strain, the increase in the acidity of lactic acid and consequentlowering of the pH value were noticed. However, with the CPN4 strain, nosignificant change in the acidity of lactic acid was noticed during thestorage period. The pH value of the fermented milk of the CPN4 strainwas changed by 0.2 during storage for two weeks, while the acidity wasincreased by 0.27% during the storage period, indicating significantsuppression of the increase in the acidic taste.

Antihypertensive Effect of the Fermented Milk on SpontaneouslyHypertensive Rat

Using the mutant strain CPN4 produced as described above, yogurt(corresponding to the culture in milk stored at 10° C. for one week inthe above-described low temperature storage test) was prepared, andorally administered by a stomachic sonde to spontaneously hypertensiverats furnished by CHARLES RIBER JAPAN INC. in order to measure thevasodepression value. As a control, milk set to the same acidity withlactic acid was employed. Each of 6 ml/kg of fermented milk andno-fermented milk (control) was forecedly administered, respectively tomale rats of 18 weeks of age (5 rats per group) and blood pressure wasmeasured after 6 hours. For measuring the blood pressure, the systolicblood pressure was found by the tail-cuff method, using an apparatus"SOFTRON BP-98A" produced by SOFTRON CO. LTD. The results are shown inTable 6.

                  TABLE 6                                                         ______________________________________                                                       Change in Systolic Blood                                       Samples        pressure after six hours                                       ______________________________________                                        Non-fermented milk                                                                           -2.8 ± 4.5 mmHg                                             CPN4 fermented milk                                                                          -25.4 ± 12.5 mmHg                                           ______________________________________                                         Vasodepression by CPN4 femented milk is significant for p<0.01 with           respect to that by nonfermented milk.                                    

From the results of Table 6, it was found that the non-fermented milkshowed no antihypertensive effect on the spontaneously hypertensiverats, while the fermented milk by the CPN4 strain showed strongantihypertensive effect on the same rats.

Example 2

To 1×10⁶ cells/ml of the CPN4 strain produced in Example 1 were addedStreptococcus thermophilus ATCC-144885 or ATCC-19258 so that eachresulting mass was 1×10⁵ cells/ml, and each resulting product was mixedand cultured at 37° C. for hours in the milk culture medium. The twocultured products were compared with each cultured product preparedusing each of the parent strain, CPN4, Streptococcus thermophilusATCC-144885 and Streptococcus thermophilus ATCC19258, respectively as tothe acidity of lactic acid, pH, peptide quantity and ACE inhibitionactivity. The results are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                                                       ACE                                                                           inhibition                                                                           Peptide                                                 Final  Acidity activity                                                                             quantity                                Strains         pH     (%)     (U/ml) (%)                                     ______________________________________                                        L. helveticis JCM1003                                                                         3.5    2.1     29.0   0.19                                    L. heleveticis CPN4                                                                           3.7    1.7     27.8   0.26                                    S. thermophilis ATTC-144885                                                                   4.5    0.7     5.0    0.06                                    S. thermophilis ATTC-19258                                                                    4.3    0.9     3.8    0.06                                    L. helveticis CPN4 +                                                                          3.9    1.2     15.0   0.19                                    S. thermophilis ATTC-144885                                                   L. helveticis CPN4 +                                                                          3.9    1.2     17.3   0.16                                    S. thermophilis ATTC-19258                                                    ______________________________________                                    

It is seen from the results of Table 7 that while the fermented milk ofthe parent strain was low in final pH and the highest in the acidity,the fermented milk of the CPN4 strain was higher than that of the parentstrain in the final pH, and was also low in the acidity. On the otherhand, the fermented milk of the Streptococcus thermophilus ATCC-144885or ATCC-19258 fermented alone, showed only negligible increase in theacidity, while being low in the peptide quantity and low in the ACEinhibition activity. If these strains were used together with the CPN4strain, the ACE inhibition activity was noticed in each of the strains,while the increase in the acidity was low. Taste was added by such mixedculturing as compared to that of the Lactobacillus helveticus as usedalone.

Although the present invention has been described with reference to thepreferred examples, it should be understood that various modificationsand variations can be easily made by those skilled in the art withoutdeparting from the spirit of the invention. Accordingly, the foregoingdisclosure should be interpreted as illustrative only and is not to beinterpreted in a limiting sense. The present invention is limited onlyby the scope of the following claims.

What is claimed is:
 1. Lactobacillus helveticus mutants induced by amutagen, and having the following specific properties:(1) an increase inacidity of 0.5% or less, said acidity resulting from an increase inlactic acid when storing a product cultured by the bacteria at 10° C.for two weeks; (2) an activity of cell membrane bound adenosinetriphosphatase being 5 μmol Pi/min/mg protein or less; and (3) thebacteria having neomycin resistance.
 2. Lactobacillus helveticus mutantsas claimed in claim 1 wherein total acidity resulting from lactic acidof the product after the storage ranges from 0.5 to 1.2%. 3.Lactobacillus helveticus mutants as claimed in claim 1 wherein thelactic acid bacteria are proliferated without extinction after culturingthe lactic acid bacteria in a neomycin-containing culture medium. 4.Lactobacillus helveticus mutants as claimed in claim 1 wherein saidbacteria have the following bacteriological properties:I. MorphologicalProperties1 ) Morphology; rod shape 2) Mobility; none 3) SporeFormation; none 4) Gram Stain; positive II. Physiological Properties:1)Catalase Production; negative 2) Indole Production; negative 3) NitrateReduction; negative 4) Aerobic Growth; facultative anaerobic 5)Formation of DL-lactic Acid from glucose by homolactic fermentationwithout formation of gases 6) Formation of Acids from Carbohydrates;glucose; + maltose; - lactose; + cellobiose; - mannose; + trehalose; -fructose; + melibiose; - galactose; + raffinose; - sucrose; -stachyose; - mannitol; - arabinose; - sorbitol; - xylose; - esculin; -rhamnose; - salicin; -7) Neomycin resistance; growth in 40 μg ofneomycin/ml or less 8) ATPase activity; 5 μmol·Pi/min/mg protein orless.
 5. Lactobacillus helveticus mutants as claimed in claim 1 whereinsaid mutagen is selected from the group consisting of UV irradiation anda chemical mutagen.